There have been rapid advances in our understanding of pattern formation in Drosophila. A number of developmental genes encode regulators that interact in a cascade of transcriptional regulation in which genes are expressed in patterns of ever increasing detail. These localized regulators control the subsequent development of morphological pattern. This proposal is motivated by the premise that an increased understanding of the regulators involved and the sites at which they act will help define the mechanisms and circuitry of this pattern-forming cascade. We have focused on homeodomain regulators because these operate at many points in the cascade, and because the extraordinary evolutionary conservation of this domain suggests that this class of regulators is universally important. In a general approach to identify and characterize functional domains in these regulators, we will use in vitro mutagenesis to create variants of engrailed and fushi tarazu and will assay function at three levels - DNA binding activity and specificity will be assayed in vitro, transcriptional regulation will be tested by transient transfection of tissue culture cells, and functional integration in the cascade of pattern formation will be examined by P-element transformation of flies. In addition to this broad screen for new functional domains, we will conduct a more directed mutational dissection of homeodomain sequences and the DNA target sequences to which they bind based on our current knowledge of the DNA binding. This analysis will assess the degree to which these homologous regulators distinguish the target sites at which they act, reveal the biochemical basis of this discrimination, and examine the importance of site discrimination to the individual roles of these regulators in the embryo. Because of the rapid progress of our knowledge of the genetic control of early pattern formation in Drosophila our biochemical dissections can be integrated in a biological perspective of growing detail.